Initial velocity methods were used to probe the kinetic mechanism of Escherichia coli uridine diphosphate-N-acetylmuramate:L-alanine ligase (UNAM:L-Ala ligase). When the activity (in the forward direction) versus substrate concentration data were plotted in double-reciprocal form, all line patterns were intersecting. The best fit of these data was to the equation for an ordered mechanism with the following parameters: k(cat), 1000 +/- 100 min(-1); Kma, 210 +/- 40 microM; Kmb, 84 +/- 20 microM; Kmc, 70 +/- 15 microM; Kia, 180 +/- 50 microM; Kib, 68 +/- 24 microM. Initial velocity line patterns were also determined when the concentration of one substrate was varied at different fixed concentrations of a second substrate while the third substrate was held at a concentration more than 100 times its Km value. Reciprocal plots of data collected with either ATP or L-alanine present at more than 100 times their Km values resulted in intersecting line patterns. Data collected with UNAM present at 100 times its Km value gave a set of parallel lines. These data are consistent with UNAM binding as the second substrate in an ordered mechanism. ADP, uridine diphosphate-N-acetylmuramoyl-L-alanine (UNAMA), and phosphate were tested as product inhibitors versus substrates. None of the products were competitive inhibitors versus L-alanine or UNAM, while the only observed competitive inhibition was ADP versus ATP. These results are consistent with an ordered kinetic mechanism wherein ATP binds first, UNAM binds second, and ADP is the last product released. Rapid quench experiments were performed in the presence of all three substrates or in the presence of ATP and UNAM. The production of acid-labile phosphate as a function of time is characterized by a burst phase followed by a slower linear phase with the rate close to k(cat) in the presence of all three substrates. Only a burst phase was observed for the time course of the reaction in the presence of ATP and UNAM. In both cases, the burst rate was identical. These observations are consistent with L-alanine being the third substrate to bind in a sequential mechanism involving a putative acyl-phosphate intermediate.

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indole-3-acetamide, carbon dioxide, and water. The kinetic mechanism of the enzyme has been determined with tryptophan as substrate at pH 8.3. Initial velocity patterns, when both amino acid and oxygen concentrations are varied, are sequential with tryptophan and ping-pong with phenylalanine and methionine. Reduction by tryptophan in the absence of oxygen is biphasic. The rate of the rapid phase varies with the tryptophan concentration, with a limiting rate of 139 s-1 and an apparent Kd value of 0.11 mM. There is a primary deuterium kinetic isotope effect on the limiting rate of reduction of 2.4. The rapid phase is followed by a slow, concentration and isotope-independent phase that is much slower than turnover; this is ascribed to dissociation of a reduced enzyme-imino acid complex. In the absence of oxygen, tryptophan is converted to indolepyruvate imine. The rate of this reaction is the same as that of the rapid phase in the reduction. Reaction of the reduced enzyme-imino acid complex with oxygen to form oxidized flavin is monophasic, with a rate constant of 196 mM-1 s-1; no intermediates are detectable. The rate of formation of indole-3-acetamide agrees with the rate of reaction with oxygen. This is followed by slow product dissociation.

pH and kinetic isotope effects on steady-state kinetic parameters have been determined for the flavoprotein tryptophan 2-monooxygenase with tryptophan, phenylalanine, 2-hydrazino-3-propanoic acid, and methionine as substrates. The V/K values of the amino acid substrates show that a residue with an apparent pKa value of 5 must be unprotonated for activity, a residue with a pKa value equal to that of the amino group of the substrate must be protonated, and deprotonation of a residue with pKa value of 10 increases the V/K value. A group in the free enzyme with a pKa value of 6 must be deprotonated for tight binding of amide inhibitors and protonated for tight binding of acids, establishing this as the intrinsic pKa value. The temperature dependence of this pKa value is consistent with involvement of a histidinyl residue. Deprotonation of the residue with a pKa value of 10 decreases binding of amide inhibitors. The D(V/Ktrp) value is less than 1.7 between pH 5 and 10, consistent with a forward commitment to catalysis of 7-15 with this substrate. The D(V/K)met value is pH dependent, increasing from a minimal value of 1.8 at pH 8.3 to a limiting value of 5.3 at both high and low pH, with pKa values of 5.1 and 10. The increase in both the isotope effect and the V/Kmet value at high pH is consistent with a conformational change to a more open active site above pH 10. The D(V/K)ala value is 5.3 at pH 8.3; this is probably the intrinsic isotope effect with this substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

Uridine diphosphate-N-acety1muramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studied included: cation specificity, ionic strength, buffer composition and pH.At 37 "C and pH 8.0, kc, was equal to 980 40 min", while K, values for ATP, UNAM, and L-alanine were, 130 ? 10, 44 ? 3, and 48 -+ 6 wM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity.Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of V,,,/K, for the three substrates and of V,,, were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 A, fl = 105", and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kc,, for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine.